Cell division must be regulated precisely, because when this regulation fails uncontrolled division and cancer ensue. During the development of multicellular organisms cell growth and division have to be coordinated with developmental signals. In addition to exit from the cell cycle to permit differentiation, a variety of cell cycle variants are utilized throughout the plant and animal kingdoms. One of these modified cell cycles, the endo cell cycle, results in DNA replication in the absence of mitosis and produces the polyploid or polytene cells that are found in at least some tissues in almost all organisms. This proposal addresses the three key aspects of how the endo cycle is regulated, using Drosophila as a model. The most significant distinction between the endo cycle and the mitotic cycle is the absence of mitosis. The morula gene is required to inhibit mitosis in the endo cycle. The MORULA protein will be identified and the mechanism by which it blocks mitosis determined. A second difference between the two cell cycles is that DNA replication is differentially regulated in the endo cycle such that some genomic regions are underreplicated while others are overreplicated and the genes amplified. Mutations in the two subunits of the E2F transcription factor, dDP and dE2F, affect differential replication during polyploid S phase. These results reveal a previously unrecognized role for E2F in controlling DNA replication once S phase has initiated. It will be determined whether E2F affects DNA replication via one of its transcriptional targets or whether it has a more direct effect on replication origins. Because the regulatory hierarchy that controls E2F activity is mutated in most human tumors, it is significant for understanding the causes of caner to determine how E2F influences DNA replication. The third level of regulation is common to both the endo cell cycle and the mitotic cycle: the transcripts that are induced at the G1-S transition must be downregulated after DNA replication has initiated in order for a subsequent round of replication to occur. The l(2)52Ec gene is unusual in being necessary both for DNA replication and downregulation of G1-S transcripts, suggesting it is a prerequisite for the cell to recognize that replication has initiated. The hypothesis that the l(2)51Ec protein is a component of the origin complex and necessary for downregulation of previously induced genes will be tested.